The development of most organs and tissues requires signal transduction pathways such as Hedgehog (Hh), TGF1, Notch, and Wnt. Most studies have focused on established components of these pathways, information flow between them, and the relatively slow transcriptional changes they drive. We propose instead a new systematic approach to discovering important rapid responses to developmental signals, using Hh signaling as a test case. Specific Aim 1: We will identify immediate-early responses to an important developmental signal, Hedgehog, applying Stable Isotope Labeling with Amino acids in Cell culture (SILAC) and mass spectrometry to developmental regulation. Specific Aim 2: We will delineate the functions of re-discovered components in the Hh immediate-early response and explore novel leads that will provide insight into signal transduction. Summary: Comprehensive assessment of the earliest responses to major developmental signals will reveal new and critical aspects of the signal transduction mechanisms. Our tests using one pathway will set the stage for applying the approach to many others.